Abstract: DNA double-strand breaks (DSBs) are rare, but highly toxic, lesions requiring orchestrated and conserved machinery to prevent adverse consequences, such as cell death and cancer-causing genome structural mutations. DSBs trigger the DNA damage response (DDR) that directs a cell to repair the break, undergo apoptosis, or become senescent. There is increasing evidence that the various endpoints of DSB processing by different cells and tissues are part of the aging phenotype, with each stage of the DDR associated with specific aging pathologies. In this Perspective, we discuss the possibility that DSBs are major drivers of intrinsic aging, highlighting the dynamics of spontaneous DSBs in relation to aging, the distinct age-related pathologies induced by DSBs, and the segmental progeroid phenotypes in humans and mice with genetic defects in DSB repair. A model is presented as to how DSBs could drive some of the basic mechanisms underlying age-related functional decline and death. DNA double-strand breaks (DSBs) are rare, but highly toxic, lesions requiring orchestrated and conserved machinery to prevent adverse consequences, such as cell death and cancer-causing genome structural mutations. DSBs trigger the DNA damage response (DDR) that directs a cell to repair the break, undergo apoptosis, or become senescent. There is increasing evidence that the various endpoints of DSB processing by different cells and tissues are part of the aging phenotype, with each stage of the DDR associated with specific aging pathologies. In this Perspective, we discuss the possibility that DSBs are major drivers of intrinsic aging, highlighting the dynamics of spontaneous DSBs in relation to aging, the distinct age-related pathologies induced by DSBs, and the segmental progeroid phenotypes in humans and mice with genetic defects in DSB repair. A model is presented as to how DSBs could drive some of the basic mechanisms underlying age-related functional decline and death. Aging can be defined as a series of time-related, degenerative processes beginning in adulthood that eventually ends life. Aging, often referred to as organismal senescence, is universal among mammals and occurs in most organisms, with some exceptions in species such as Hydra. Aging leads to loss of function and a steeply increasing risk of diseases, such as cancer, heart disease, and diabetes. While multifactorial in nature, it is conceivable that aging is ultimately caused by a combination of toxic by-products of normal metabolism, such as reactive oxygen species (ROS) and imperfections in the systems that normally are capable of repairing cellular damage. Because of its central role in all living systems, DNA has long been considered a major target of age-related cellular damage and DNA damage a potential universal cause of aging (Gorbunova and Seluanov, 2016Gorbunova V. Seluanov A. DNA double strand break repair, aging and the chromatin connection.Mutat. Res. 2016; 788: 2-6Crossref PubMed Scopus (54) Google Scholar, Vijg, 2007Vijg J. Aging of the Genome: the Dual Role of the DNA in Life and Death. Oxford University Press, 2007Crossref Scopus (35) Google Scholar). Indeed, the challenge that such damage posed to even the earliest living systems necessitated the emergence of highly advanced, conserved pathways of genome maintenance. Without such evolved pathways, cells could not survive the high influx of spontaneous DNA damage, which is estimated at ∼2 × 105 lesions per day in a typical mammalian cell (Barnes and Lindahl, 2004Barnes D.E. Lindahl T. Repair and genetic consequences of endogenous DNA base damage in mammalian cells.Annu. Rev. Genet. 2004; 38: 445-476Crossref PubMed Scopus (621) Google Scholar). The majority of this damage is repaired swiftly and therefore unlikely to drive the aging process; however, some highly toxic lesions persisting in a very small quantity can pose a considerable threat to the cell and the organism. The best known of such lesions is the DNA double-strand break (DSB). DNA DSBs occur in any given cell in the order of 10 to 50 per cell per day, depending on cell cycle and tissue (Vilenchik and Knudson, 2003Vilenchik M.M. Knudson A.G. Endogenous DNA double-strand breaks: production, fidelity of repair, and induction of cancer.Proc. Natl. Acad. Sci. USA. 2003; 100: 12871-12876Crossref PubMed Scopus (491) Google Scholar). DSBs, as well as other lesions affecting both DNA strands, such as interstrand cross-links (ICLs), are difficult to remove and often lead to cell death, cellular senescence, or mutations (most notably genome structural variation). DSBs can be induced spontaneously, for example, by ROS, but also through exposure to ionizing radiation, treatment with chemotherapeutic agents, or even through ingestion of nutritional supplements, such as bioflavonoids. In this Perspective, we will review current evidence that DNA DSBs are key lesions driving some of the specific molecular and cellular end points that characterize mammalian aging and age-related disease. Once a DSB occurs within a cell, there is a rapid, concerted signaling cascade to process the damage and prevent adverse consequences that could impact cellular function and survival. Collectively, this is known as the DNA damage response (DDR), which has been extensively reviewed, both for DNA damage in general and specifically for DSBs (Ceccaldi et al., 2016Ceccaldi R. Rondinelli B. D’Andrea A.D. Repair pathway choices and consequences at the double-strand break.Trends Cell Biol. 2016; 26: 52-64Abstract Full Text Full Text PDF PubMed Scopus (842) Google Scholar, Ciccia and Elledge, 2010Ciccia A. Elledge S.J. The DNA damage response: making it safe to play with knives.Mol. Cell. 2010; 40: 179-204Abstract Full Text Full Text PDF PubMed Scopus (2925) Google Scholar, Jackson and Bartek, 2009Jackson S.P. Bartek J. The DNA-damage response in human biology and disease.Nature. 2009; 461: 1071-1078Crossref PubMed Scopus (3828) Google Scholar). Here, the DDR in response to DSBs will be discussed in terms of its possible relationship with aging and longevity. DSBs are detected by sensor proteins, most notably the MRE11/RAD50/NBS1 (MRN) complex and the Ku70/Ku80 heterodimer, which through many possible mediators and effectors, signal the processes that lead to a number of possible outcomes, almost all of which are relevant to the aging process. The pathway choice that determines the outcome depends on a number of factors, including the type and/or severity of the damage, the cell type (e.g., mitotically active or not, stem cell or normal somatic cell), cell-cycle stage, chromatin status, and, possibly, age of the organism (Ceccaldi et al., 2016Ceccaldi R. Rondinelli B. D’Andrea A.D. Repair pathway choices and consequences at the double-strand break.Trends Cell Biol. 2016; 26: 52-64Abstract Full Text Full Text PDF PubMed Scopus (842) Google Scholar). The best possible outcome of DSB processing is actual repair and restoration of an intact double helix. However, even if repair is successfully completed, it may have been erroneous, resulting in DNA mutations. Other pathway choices with possible adverse outcomes relevant to aging are apoptosis and cellular senescence. At early age, under normal physiological conditions, adverse outcomes of DSB processing are rare and not immediately harmful to the organism. Indeed, they are beneficial since rapid repair promotes cell survival, even if this occasionally goes at the expense of mutations. The same applies to apoptosis, generally an efficient way to cleanse the soma of irreversibly damaged cells. Likewise, cellular senescence is now established as a key developmental process (Muñoz-Espín et al., 2013Muñoz-Espín D. Cañamero M. Maraver A. Gómez-López G. Contreras J. Murillo-Cuesta S. Rodríguez-Baeza A. Varela-Nieto I. Ruberte J. Collado M. Serrano M. Programmed cell senescence during mammalian embryonic development.Cell. 2013; 155: 1104-1118Abstract Full Text Full Text PDF PubMed Scopus (835) Google Scholar), as well as a trigger of tissue remodeling upon damage (Demaria et al., 2014Demaria M. Ohtani N. Youssef S.A. Rodier F. Toussaint W. Mitchell J.R. Laberge R.M. Vijg J. Van Steeg H. Dollé M.E. et al.An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA.Dev. Cell. 2014; 31: 722-733Abstract Full Text Full Text PDF PubMed Scopus (993) Google Scholar). However, at old age, the cumulative effects of all these events may well be a cause of some of the well-documented phenotypes of aging, such as atrophy, inflammation, immuno-senescence, and cancer. One of the first questions arising in relation to DSBs and aging is whether the frequency of such events increases with age. Spontaneous DNA damage levels in human and animal tissues are normally very low, in spite of their high influx, due to the enormous efficiency of genome maintenance, making it difficult to study these events during normal aging in mammals. This is especially true for the highly toxic DSBs. However, snapshot estimates of DSB levels can be inferred by detecting DSB protein aggregates as microscopically visible, subnuclear foci. Most commonly used is phosphorylated histone H2AX (γH2AX), which often co-localizes with ataxia telangiectasia mutated (ATM), 53BP1, and RAD51. γH2AX foci are often used as surrogates for DSBs based on their quantitative correlation with increasing doses of ionizing radiation (Rothkamm et al., 2015Rothkamm K. Barnard S. Moquet J. Ellender M. Rana Z. Burdak-Rothkamm S. DNA damage foci: meaning and significance.Environ. Mol. Mutagen. 2015; 56: 491-504Crossref PubMed Scopus (192) Google Scholar). Interestingly, while most foci quickly disappear after radiation, some remain for days to months (Siddiqui et al., 2015Siddiqui M.S. François M. Fenech M.F. Leifert W.R. Persistent γH2AX: a promising molecular marker of DNA damage and aging.Mutat. Res. Rev. Mutat. Res. 2015; 766: 1-19Crossref PubMed Scopus (124) Google Scholar). γH2AX foci were especially shown to be highly persistent in close proximity to telomeres (Fumagalli et al., 2012Fumagalli M. Rossiello F. Clerici M. Barozzi S. 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While such persistent γH2AX foci could reflect insufficient H2AX dephosphorylation (Mamouni et al., 2014Mamouni K. Cristini A. Guirouilh-Barbat J. Monferran S. Lemarié A. Faye J.C. Lopez B.S. Favre G. Sordet O. RhoB promotes γH2AX dephosphorylation and DNA double-strand break repair.Mol. Cell. Biol. 2014; 34: 3144-3155Crossref PubMed Scopus (29) Google Scholar), which may get worse with age, it is tempting to speculate that instead they mark DSBs that are difficult to repair, such as uncapped telomeres, which could explain their accumulation over time. Since persistency of γH2AX foci appears to depend on genomic location, it would be important to analyze DSBs with more direct assays capable of mapping these events genome wide at the base pair level (Crosetto et al., 2013Crosetto N. Mitra A. Silva M.J. Bienko M. Dojer N. Wang Q. Karaca E. Chiarle R. Skrzypczak M. Ginalski K. et al.Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing.Nat. Methods. 2013; 10: 361-365Crossref PubMed Scopus (311) Google Scholar). Of note, the observed accumulation of persistent γH2AX foci is in keeping with a wealth of evidence that increased macromolecular damage, possibly as a consequence of imperfect maintenance systems, is a hallmark of the aging process (López-Otín et al., 2013López-Otín C. Blasco M.A. Partridge L. Serrano M. Kroemer G. The hallmarks of aging.Cell. 2013; 153: 1194-1217Abstract Full Text Full Text PDF PubMed Scopus (7746) Google Scholar). The presence of persistent DSBs has been shown to directly affect the functionality of the cell, specifically within the stem cell compartment. For example, when quiescent, HSCs are not affected by DSBs; however, when prompted to replicate in response to stress, HSCs from aged mice were found to divide more slowly or die (Flach et al., 2014Flach J. Bakker S.T. Mohrin M. Conroy P.C. Pietras E.M. Reynaud D. Alvarez S. Diolaiti M.E. Ugarte F. 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Kaschutnig P. et al.Exit from dormancy provokes DNA-damage-induced attrition in haematopoietic stem cells.Nature. 2015; 520: 549-552Crossref PubMed Scopus (393) Google Scholar). Indeed, replication stress has been identified as a potent driver of functional decline of old HSCs (Flach et al., 2014Flach J. Bakker S.T. Mohrin M. Conroy P.C. Pietras E.M. Reynaud D. Alvarez S. Diolaiti M.E. Ugarte F. Forsberg E.C. et al.Replication stress is a potent driver of functional decline in ageing haematopoietic stem cells.Nature. 2014; 512: 198-202Crossref PubMed Scopus (408) Google Scholar). Proliferating stem cells are very sensitive to radiation-induced apoptosis, which then forces quiescent stem cells to proliferate to replace lost stem cells. Interestingly, when HSCs exit from quiescence, virtually all DNA repair pathways are stimulated to repair damage before the cells continue cycling, even in aged HSC populations. The latter were found to exhibit a larger amount of single-strand breaks (SSBs) and DSBs than cells from young animals (Beerman et al., 2014Beerman I. Seita J. Inlay M.A. Weissman I.L. Rossi D.J. Quiescent hematopoietic stem cells accumulate DNA damage during aging that is repaired upon entry into cell cycle.Cell Stem Cell. 2014; 15: 37-50Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). Hence, at least in the hematopoietic system, DSBs limit cell renewal capacity, and accumulation of DSBs during aging could adversely affect regeneration. In the neuronal stem cell compartment, the effects of DSBs are different. Here, when neuronal stem cells encounter damage, they are forced to undergo premature senescence or terminally differentiate into astroglial cells (Schneider et al., 2013Schneider L. Pellegatta S. Favaro R. Pisati F. Roncaglia P. Testa G. Nicolis S.K. Finocchiaro G. d’Adda di Fagagna F. DNA damage in mammalian neural stem cells leads to astrocytic differentiation mediated by BMP2 signaling through JAK-STAT.Stem Cell Reports. 2013; 1: 123-138Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). This forced senescence intrinsically diminishes the proliferative potential of neuronal stem cells and over time may contribute to the neurodegeneration and cognitive impairment often seen in aging. Taken together, there is consensus that γH2AX foci increase with age in multiple tissue types, as well as in senescent cells. It is unknown whether this increase reflects the accumulation of unrepaired DSBs, progressively delayed repair events, possibly as a consequence of an inherently limited capacity to process DSBs, or, simply, increasingly ineffective dephosphorylation. The latter seems unlikely because age-related accumulation of 53BP1 foci, an independent marker for DSB repair, has also been observed (White et al., 2015White R.R. Milholland B. de Bruin A. Curran S. Laberge R.M. van Steeg H. Campisi J. Maslov A.Y. Vijg J. Controlled induction of DNA double-strand breaks in the mouse liver induces features of tissue ageing.Nat. Commun. 2015; 6: 6790Crossref PubMed Scopus (69) Google Scholar). While the data pointing to possible physiological effects of γH2AX foci accumulating with age, specifically in HSCs, still remain inconsistent and need further study, it is reasonable to hypothesize that DSB accumulation during aging is a major factor in the impairment of stem cell proliferative capacity. Based on the evidence that unrepaired DSBs accumulate with age and are associated with a major age-related cellular phenotype, i.e., cellular senescence, it is important to study various aspects of the DDR in relation to aging. Interestingly, levels of ATM and its phosphorylation after ionizing radiation, which determines the kinase activity, were found to decrease in aged mice. This decreased ATM function was suggested to underlie the observed decline in p53 response to ionizing radiation in old animals (Feng et al., 2007Feng Z. Hu W. Teresky A.K. Hernando E. Cordon-Cardo C. Levine A.J. Declining p53 function in the aging process: a possible mechanism for the increased tumor incidence in older populations.Proc. Natl. Acad. Sci. USA. 2007; 104: 16633-16638Crossref PubMed Scopus (204) Google Scholar). Also, the increased level of persistent γH2AX foci in ovarian mouse and human primordial follicles mentioned above was found to be accompanied by a reduced expression level of genes involved in DSB processing, including ATM, MRE11, RAD51, and BRCA1, but not BRCA2 (Oktay et al., 2015Oktay K. Turan V. Titus S. Stobezki R. Liu L. BRCA mutations, DNA repair deficiency, and ovarian aging.Biol. Reprod. 2015; 93: 67Crossref PubMed Scopus (98) Google Scholar, Titus et al., 2013Titus S. Li F. Stobezki R. Akula K. Unsal E. Jeong K. Dickler M. Robson M. Moy F. Goswami S. Oktay K. Impairment of BRCA1-related DNA double-strand break repair leads to ovarian aging in mice and humans.Sci. Transl. Med. 2013; 5: 172ra21Crossref PubMed Scopus (313) Google Scholar), which may account for the decline of oocyte reserves in aging mammals. More information is available on the capacity to repair DSBs in relation to age. DSB repair consists of two main pathways: homologous recombination (HR) and non-homologous end joining (NHEJ) (Aparicio et al., 2014Aparicio T. Baer R. Gautier J. DNA double-strand break repair pathway choice and cancer.DNA Repair (Amst.). 2014; 19: 169-175Crossref PubMed Scopus (221) Google Scholar, Chapman et al., 2012Chapman J.R. Taylor M.R. Boulton S.J. Playing the end game: DNA double-strand break repair pathway choice.Mol. Cell. 2012; 47: 497-510Abstract Full Text Full Text PDF PubMed Scopus (1102) Google Scholar). In mammals, NHEJ is the most commonly used pathway in all phases of the cell cycle. It does not require homologous ends and signals DSBs through the Ku70/Ku80 heterodimer and DNA-PKcs. After minimal processing by nucleases, the two ends of broken DNA are joined by the LIGIV/XRCC4/XLF complex. NHEJ is error prone because end processing can lead to small base pair deletions and translocations (Ghezraoui et al., 2014Ghezraoui H. Piganeau M. Renouf B. Renaud J.B. Sallmyr A. Ruis B. Oh S. Tomkinson A.E. Hendrickson E.A. Giovannangeli C. et al.Chromosomal translocations in human cells are generated by canonical nonhomologous end-joining.Mol. Cell. 2014; 55: 829-842Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, Lieber, 2010Lieber M.R. The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway.Annu. Rev. Biochem. 2010; 79: 181-211Crossref PubMed Scopus (1923) Google Scholar). In addition to classical NHEJ, alternate pathways, such as Alt-NHEJ or microhomology-mediated end joining (MMEJ), can operate in mammalian cells. Although distinct mechanisms between Alt-NHEJ and MMEJ are still an active area of research, in general, both are kinetically slower than NHEJ, rely on microhomologies at DNA ends, and can give rise to larger base pair deletions than classical NHEJ, as well as translocations. Indeed, these alternative pathways are now considered a major source of genomic instability, which makes them highly relevant as DSB-induced mechanisms of aging and senescence. The alternative end-joining pathways also involve different proteins; they can be initiated by PARP1 and can also involve factors that function in HR, such as BRCA1 (Iliakis et al., 2015Iliakis G. Murmann T. Soni A. Alternative end-joining repair pathways are the ultimate backup for abrogated classical non-homologous end-joining and homologous recombination repair: Implications for the formation of chromosome translocations.Mutat. Res. Genet. Toxicol. Environ. 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While generally error free, HR can still give rise to genome rearrangements when recombination involves homologous sequences on heterologous chromosomes or repeat elements (Hu et al., 2013Hu L. Kim T.M. Son M.Y. Kim S.A. Holland C.L. Tateishi S. Kim D.H. Yew P.R. Montagna C. Dumitrache L.C. Hasty P. Two replication fork maintenance pathways fuse inverted repeats to rearrange chromosomes.Nature. 2013; 501: 569-572Crossref PubMed Scopus (35) Google Scholar, Morales et al., 2015Morales M.E. White T.B. Streva V.A. DeFreece C.B. Hedges D.J. Deininger P.L. The contribution of alu elements to mutagenic DNA double-strand break repair.PLoS Genet. 2015; 11: e1005016Crossref PubMed Scopus (52) Google Scholar, Richardson and Jasin, 2000Richardson C. Jasin M. Frequent chromosomal translocations induced by DNA double-strand breaks.Nature. 2000; 405: 697-700Crossref PubMed Scopus (421) Google Scholar). For example, in yeast, unequal homologous recombination at the rDNA locus leads to the production of extrachromosomal DNA, which is likely to be the cause of yeast replicative aging (Sinclair and Guarente, 1997Sinclair D.A. Guarente L. Extrachromosomal rDNA circles—a cause of aging in yeast.Cell. 1997; 91: 1033-1042Abstract Full Text Full Text PDF PubMed Scopus (1173) Google Scholar), but can also occur in humans (Warmerdam et al., 2016Warmerdam D.O. van den Berg J. Medema R.H. Breaks in the 45S rDNA lead to recombination-mediated loss of repeats.Cell Rep. 2016; 14: 2519-2527Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). While rapid joining of DSB ends is obviously beneficial for the survival of the cell, the mutations associated with DSB repair are potential causal factors in aging and age-related diseases, most notably cancer. Interestingly, based on the known frequency of cytogenetically detectable chromosome breaks in human cells, it has been estimated that over the lifetime of an average individual, each typical somatic cell would have an estimated 2,300 imprecise repair sites distributed throughout the genome as a consequence of errors associated with end joining (Lieber and Karanjawala, 2004Lieber M.R. Karanjawala Z.E. Ageing, repetitive genomes and DNA damage.Nat. Rev. Mol. Cell Biol. 2004; 5: 69-75Crossref PubMed Scopus (102) Google Scholar). To confirm this estimate, it would be necessary to analyze the somatic genome for all possible genome structural variations, including deletions varying from a few to many thousands of base pairs. Since such events would be different from cell to cell, such an analysis has not been possible. However, genome structural variations, including deletions and translocations, have been demonstrated to occur in mouse somatic tissues and increase with age (Dollé et al., 1997Dollé M.E. Giese H. Hopkins C.L. Martus H.J. Hausdorff J.M. Vijg J. Rapid accumulation of genome rearrangements in liver but not in brain of old mice.Nat. Genet. 1997; 17: 431-434Crossref PubMed Scopus (214) Google Scholar, Dollé et al., 2000Dollé M.E. Snyder W.K. Gossen J.A. Lohman P.H. Vijg J. Distinct spectra of somatic mutations accumulated with age in mouse heart and small intestine.Proc. Natl. Acad. Sci. USA. 2000; 97: 8403-8408Crossref PubMed Scopus (201) Google Scholar). These results were based on breakpoints detected in a genomically integrated selectable reporter gene rather than genome-wide sequence analysis. By extrapolating from the 3,000 bp reporter locus to the genome overall, genome rearrangements in the mouse heart were estimated to increase with age to about 40 rearrangements per cardiomyocyte (Dollé and Vijg, 2002Dollé M.E. Vijg J. Genome dynamics in aging mice.Genome Res. 2002; 12: 1732-1738Crossref PubMed Scopus (59) Google Scholar). More recently, quantitative assays have been developed to comprehensively analyze genome structural variation by next-generation sequencing (Chiarle et al., 2011Chiarle R. Zhang Y. Frock R.L. Lewis S.M. Molinie B. Ho Y.J. Myers D.R. Choi V.W. Compagno M. Malkin D.J. et al.Genome-wide translocation sequencing reveals mechanisms of chromosome breaks and rearrangements in B cells.Cell. 2011; 147: 107-119Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar, Maslov et al., 2015Maslov A.Y. Quispe-Tintaya W. Gorbacheva T. White R.R. Vijg J. High-throughput sequencing in mutation detection: a new generation of genotoxicity tests?.Mutat. 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The first evidence of decreased NHEJ efficiency in aging was observed in senescent human diploid fibroblasts, where NHEJ events were scored using a GFP reporter plasmid co-transfected with I-SceI, a restriction enzyme, to induce DSBs within the construct (Seluanov et al., 2004Seluanov A. Mittelman D. Pereira-Smith O.M. Wilson J.H. Gorbunova V. DNA end joining becomes less efficient and more error-prone during cellular senescence.Proc. Natl. Acad. Sci. USA. 2004; 101: 7624-7629Crossref PubMed Scopus (216) Google Scholar). However, cellular senescence, i.e., in vitro aging, is not the same as organismal aging. Nevertheless, these findings were consistent with results showing that the capacity of cell extracts prepared from isolated neurons from rat cerebral cortex to join a linearized plasmid declines with age (Vyjayanti and Rao, 2006Vyjayanti V.N. Rao K.S. DNA double-strand break repair in brain: reduced NHEJ activity in aging rat neurons.Neurosci. 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